1. Field of the Invention
This invention concerns methods for detection of Cryptosporidium species or individual Cryptosporidium isolates and for diagnosis of prior or concurrent Cryptosporidium infections. The method for detection of Cryptosporidium species involves detection of a Cryptosporidium surface antigens GP900, p68 or cryptopain, GP900, p68 or cryptopain antibodies, and GP900, p68 or cryptopain DNAs and RNAs using PCR primers from regions flanking different domains of GP900, p68 or cryptopain. The method for detection of Cryptosporidium isolates involves PCR amplification of portions of GP900, P68, cryptopain and flanking regions with or without restriction fragment length polymorphism analysis which yield a fingerprint for each individual isolate. The method for diagnosis of Cryptosporidium infections involves detecting a presence of GP900, p68 or cryptopain antibodies, GP900, p68 or cryptopain antigen or the DNA or RNA encoding the GP900, p68 or cryptopain antigen in biological and environmental samples. The methods of the invention detect the Cryptosporidium antigen comprised of a protein with or without carbohydrates attached thereto, or the DNA or RNA encoding the Cryptosporidium antigen or DNA adjacent to it (flanking regions), or a mutant, variant, analog or fragment thereof. The invention additionally concerns methods for production of recombinant Cryptosporidium antigens suitable for development of diagnostic and detection tools and kits.
Cryptosporidium is an Apicomlexan protozoa which causes gastrointestinal disease in humans and other vertebrates. In immunocompetent host, the disease process ends when protective antibody develops. In immunocompromised hosts, the disease may become chronic resulting in wasting, diarrhea, electrolyte abnormalities, dehydration and death. There is no effective treatment for cryptosporidiosis.
The genus Cryptosporidium consists of Apicomplexan parasites that invade and develop within epithelial cells of the gastrointestinal, hepatobiliary and respiratory tracts of a wide variety of vertebrates including reptiles, birds and mammals. Cryptosporidium was recognized as a cause of animal disease for several decades before the first cases of human cryptosporidiosis were reported in 1976. However, it was not until 1982 that the magnitude of disease caused by this parasite in both AIDS patients and immunocompetent hosts began to be appreciated. Subsequently, Cryptosporidium has been found to be one of the most common causes of human diarrhea worldwide, and to be an increasingly recognized cause of diarrhea in children, animal care workers, and travelers. (Cryptosporidium and Cryptosporidiosis in Humans, Ed. Fayer, R., CRC Press, Boca Raton (1997)).
Large water-borne outbreaks of cryptosporidiosis caused by contaminated municipal water supplies in the US or in the UK have been noted in the last decade (N. Engl. J. Med., 320:1372 (1989), and 33:161 (1994)). A large outbreak in Milwaukee in April 1993 involved 400,000 persons and led to the subsequent deaths of more than hundred immunocompromised persons. Like a number of other waterborne outbreaks, the Milwaukee outbreak appears to have been due to contamination from farm or abattoir run-off and was specifically connected to cryptosporidiosis infected cows and calves. Nosocomial transmission in hospitals from patients to staff, patient to patient, and contaminated ice to patients and staff have also been well documented (J. Infect. Dis., 158:647 (1985)).
Waterborne and nosocomial spread uncovered a number of biological characteristics of oocysts. First, the infectious dose of a parasite is very low. The ID50 for human volunteers with normal immune systems is 132 oocysts (N. Engl. J. Med., 332:855 (1995)). Second, infected hosts, for example calves, excrete large numbers of oocysts, on the order of 1010/day. Third, the oocysts are fully sporulated and ready to infect when excreted. Fourth, the oocysts are environmentally hardy. They remain infectious in cool, moist areas for 3-4 months and are not killed by chlorine levels achievable in drinking water. Fifth, the oocysts are quite small, 4-6 xcexcm, and are thus difficult to filter.
The infective forms of Cryptosporidium, called sporozoites and merozoites, appear to adhere to the host cell and release the contents of anterior organelles (rhoptries, micronemes or dense granules) during the invasion process (Parasitol. Today, 8:28(1992)). Proteins involved in these events have in many instances been found to be the target of invasion blocking immunity in vitro and neutralization in vivo (Infect. Immun., 56:2538(1988)).
While the actual interaction between Cryptosporidium and the host""s immune system is poorly understood, it is known that disruption of either the cellular or the humoral components can result in protracted cryptosporidiosis (Parasitol. Today, 8:24 (1992)). Specific antibodies alone appear to be enough to neutralize the organism""s infectivity. In vitro and in vivo observations indicate that antibodies to Cryptosporidium parvum inhibit invasion and intracellular development leading to protection in challenge experiments, or amelioration of infection in established disease (Infect. Immun., 59:1172 (1991)).
One source of such antibodies is hyperimmune bovine colostrum (HBC) collected from cows immunized with Cryptosporidium oocysts. Calves challenged with Cryptosporidium oocysts are protected from the development of disease by the administration of HBC (Infect. Immun., 61:4079 (1993)). Some immunocompromised AIDS patients infected with Cryptosporidium have also responded to HBC with a reduction in or disappearance of the symptoms of the disease (Gastroenterology, 98:486 (1990)). Immunoglobulin from HBC (HBC Ig) has been found to inhibit the ability of the sporozoite to invade and/or develop intracellularly in vitro and it has been used to immunoprecipitate at least 22 different surface radioiodinated proteins of Cryptosporidium sporozoites. Western blot analysis of proteins of whole oocysts which contain sporozoite indicates that HBC predominantly recognizes two proteins of sizes 250 KD and  greater than 900 KD (Infect. Immun., 61:4079 (1993)).
Although a connection between Cryptosporidium from water, dairy animals, pets, children in day care and hospital environments and cryptosporidiosis has been made, the relative importance of water, food, pets, sexual or casual person-to-person contact in the transmission of the parasite has not been established. The epidemiology of Cryptosporidium, particularly transmission and reservoirs of the parasite have been difficult to study because the organism cannot be propagated in vitro for the development of serological or growth characteristics as a method of systematic identification of Cryptosporidium species and individual isolates.
Therefore, availability of Cryptosporidium specific antigen, DNA and antibody markers for species identification and for differentiation of strains/isolates within species would be greatly advantageous.
While, as described above, the Cryptosporidium infection can have serious and, in some cases, fatal consequences, only limited detection and diagnostic methods, tools and kits are available.
Currently available Cryptosporidium detection methods are microbiological immunological assays and limited PCR-based detections. The microbiological detections of the organism in the stool has a very low sensitivity of detection of about 1000 organisms in one gram of stool. Sensitivity of antibody detection methods is somewhat improved but these methods still can detect the presence of the organism only when at least 500 organisms are present in one gram of stool. Attempts to use monoclonal antibodies for detection of cryptosporidiosis resulted in very expensive and not overly sensitive kits which, so far, have not generally replaced the classical microbiological detection techniques.
Thus, better and more sensitive methods for detection of Cryptosporidium are needed.
It is a primary objective of this invention to provide methods for detection of Cryptosporidium species or isolates by 1) specific detection of GP900, P68 or cryptopain antigens, 2) specific detection of GP900, P68 or cryptopain DNA fragments, and 3) detection of the presence of anti-Cryptosporidium antibodies indicating that the host has been, in the past, or is currently infected with Cryptosporidium.
All patents, patent applications and publications cited herein are hereby incorporated by reference.
One aspect of this invention concerns methods and kits for detection of Cryptosporidium species or individual Cryptosporidium isolates and for diagnosis of prior or concurrent Cryptosporidium infections.
Another aspect of this invention concerns a method for detection of Cryptosporidium species involving detection of a Cryptosporidium antigen GP900, p68 or cryptopain, GP900, p68 or cryptopain antibodies, and GP900, p68 or cryptopain DNAs and RNAs using PCR primers from regions flanking different domains of GP900, p68 or cryptopain.
Still another aspect of this invention concerns a method for detection of Cryptosporidium isolates involving PCR amplification of DNA fragments which are different in different isolates, such as poly-threonine rich domains or different restriction sites which yield a fingerprint for each individual isolate differentiating it from another isolate.
Yet another aspect of this invention concerns a method for diagnosis of Cryptosporidium infections involving detection of the presence of GP900, p68 or cryptopain antibodies, GP900, p68 or cryptopain antigen or the DNA or RNA encoding the GP900, p68 or cryptopain antigen protein with or without carbohydrates attached thereto, or a mutant, variant, analog or fragment thereof in biological and environmental samples.
Still yet another aspect of this invention concerns methods for production of recombinant Cryptosporidium antigens suitable for development of diagnostic and detection tools and kits.
Another aspect of this invention concerns antibodies, antigens, DNAs and RNAs used in methods or kits for detection of Cryptosporidium species and isolates or diagnosis of prior or concurrent Cryptosporidium infections.
Another aspect of this invention concerns polyclonal or monoclonal antibodies directed against the Cryptosporidium antigen for use in a method and kits for detection of cryptosporidiosis.
Another aspect of this invention concerns the GP900, p68 or cryptopain Cryptosporidium antigen or fragments thereof.
Still another aspect of this invention concerns a DNA and RNA encoding the Cryptosporidium antigen and fragments thereof suitable for preparation of anti-Cryptosporidium antibodies.
Still yet another aspect of the invention is the use of the GP900, p68 or cryptopain antigen, antibody, DNA or RNA for Cryptosporidium diagnosis of prior or current infection in a human or animal host or for detection of Cryptosporidium parasite in the environment.
Still another aspect of this invention concerns a DNA and RNA encoding the Cryptosporidium protein or glycoprotein comprising Cryptosporidium antigen or fragments thereof for use in production of the protein or glycoprotein for development of agents used for diagnosis or detection of Cryptosporidium infection.
Another aspect of this invention concerns the DNA sequence of 7334 bp (SEQ ID NO: 1) nucleotides encoding the GP900 protein of Iowa isolate and its upstream (5xe2x80x2) protein coding and regulatory elements and its 3xe2x80x2 noncoding sequence.
Another aspect of this invention concerns the DNA sequence of 5511 bp (SEQ ID NO: 2) nucleotides encoding GP900 of the Iowa isolate, and its nucleotide and size variants.
Another aspect of this invention concerns an amino acid sequence of 1837 amino acids (SEQ ID NO: 5) of GP900 of Iowa isolate, a  greater than 900 kD glycoprotein present in or on the surface of sporozoites and merozoites, and its amino acid and size variants.
Another aspect of this invention concerns the DNA sequence of 5318 bp (SEQ ID NO: 3) nucleotides encoding the GP900 protein of NINC isolate comprised of partial ORF and 3xe2x80x2 flanking region.
Another aspect of this invention concerns the DNA sequence of 5163 bp (SEQ ID NO: 4) nucleotides encoding GP900 of the NINC isolate comprised of the partial ORF.
Another aspect of this invention concerns an amino acid sequence of 1721 amino acids (SEQ ID NO: 6) of GP900 of NINC isolate.
Another aspect of this invention concerns the DNA sequence of 2380 nucleotides (SEQ ID NO: 25) encoding a protein portion of P68, its nucleotide and size variants and its upstream (5xe2x80x2) protein coding and regulatory elements.
Another aspect of this invention concerns an amino acid sequence comprising 503 amino acids (SEQ ID NO: 26) of a protein portion of P68, a 50-100 kDa glycoprotein of sporozoites and merozoites, and its amino acid and size variants.
Another aspect of this invention concerns the DNA sequence of 1663 bp (SEQ ID NO: 27) of cryptopain comprised of 5xe2x80x2 and 3xe2x80x2 flanking sequences, its pre pro fragments (SEQ ID NO: 28) and mature enzyme (SEQ ID NO: 29).
Another aspect of this invention concerns an amino acid sequence of 401 amino acids (SEQ ID NO: 30) of cryptopain, cryptopain pre and pro fragments (SEQ ID NO: 31), and mature enzyme (SEQ ID NO: 32).
Still another aspect of this invention are PCR primers which are oligonucleotides of about 14 to about 35 bp in length synthesized using portions of the GP900, P68 or cryptopain DNA and which are used by enzymatic reactions leading to the amplification of DNA.
Yet another aspect of this invention is a DNA probe which is an oligonucleotide or a larger segment of DNA used for direct hybridization with complementary DNA or RNA sequences.
Still yet another aspect of this invention concerns a method for diagnosing Cryptosporidium infection of a subject, comprising steps:
(a) contacting a body specimen, fluid or tissue obtained from the subject with an anti-Cryptosporidium monoclonal or polyclonal antibody; and
(b) detecting the formation of antibody-antigen complex wherein the presence of the complex indicates positive diagnosis of the presence of a Cryptosporidium organism in the subject and wherein the absence of the complex indicates negative diagnosis of Cryptosporidium infection.
Still yet another aspect of this invention concerns a method for detecting a presence of Cryptosporidium parasite in a biological or environmental sample, said method comprising steps:
(a) contacting a biological or environmental sample with an anti-Cryptosporidium monoclonal or polyclonal antibody; and
(b) detecting the formation of antibody-antigen complex wherein the presence of the complex indicates presence of the Cryptosporidium parasite in the sample and wherein the absence of the complex indicates absence of Cryptosporidium parasite in the sample.
Still yet another aspect of this invention concerns a method for detecting anti-Cryptosporidium antibody in a subject, said method comprising steps:
(a) contacting a body specimen, fluid or tissue obtained from the subject with the GP900, p68 or cryptopain antigen; and
(b) detecting a formation of antibody-antigen complex wherein the presence of the complex indicates positive diagnosis of the presence of a Cryptosporidium antibody in the subject and wherein the absence of the complex indicates negative diagnosis of Cryptosporidium infection.
Still another aspect of this invention is a Cryptosporidium diagnostic or detection kit comprising anti-Cryptosporidium specific monoclonal and polyclonal antibodies, Cryptosporidium GP900, P68 or cryptopain antigen according to the invention and a means for detection of an antibody-antigen complex.